目的 通过研究柴参解郁汤对腹泻为主型肠易激综合征(Diarrhea predominant irritable bowel syndrome,IBS-D)模型小鼠粪便中胆汁酸代谢轮廓的影响,探讨柴参解郁汤改善IBS-D的可能机制.方法 将C57BL/6小鼠随机分为3组,空白组、模型组、柴参解郁汤组(15 g·kg-1).IBS-D小鼠模型采用束缚性应激复合灌胃番泻叶造模,造模成功后,灌胃给予柴参解郁汤2周,分析小鼠粪便含水量,检测空肠病理损伤,Western blot检测空肠中COX-2、iNOS的蛋白表达,通过检测FITC标记的右旋糖苷及紧密连接蛋白ZO-1、OCCLUDIN的mRNA表达,测定小鼠胃肠道通透性.进一步采用靶向代谢组学分析小鼠粪便胆汁酸代谢轮廓.结果 与空白组相比,模型组小鼠粪便含水量增高(P<0.01),给予柴参解郁汤后粪便含水量下降(P<0.01);与空白组相比,模型组小鼠空肠中COX-2和iNOS蛋白表达量上调(P<0.05,P<0.01),而柴参解郁汤可下调空肠细胞中iNOS蛋白表达(P<0.01);与空白组相比,模型组小鼠空肠中ZO-1、OCCLUDIN的mRNA表达量明显下降(P<0.01),FIFC-右旋糖苷含量下降(P<0.01),柴参解郁汤治疗后表达量有所回调(P<0.05,P<0.01).与空白组相比,模型组小鼠空肠绒毛大部分脱落坏死,绒毛的固有层细胞溶解,绒毛结构模糊不清,给予柴参解郁汤后趋于正常.IBS-D小鼠粪便中胆汁酸组成和含量发生显著改变,柴参解郁汤可显著回调模型组小鼠的胆汁酸代谢紊乱,如石胆酸(LCA)、去氧胆酸(DCA)、鹅去氧胆酸(CDCA)等.结论 柴参解郁汤可有效缓解IBS-D小鼠空肠组织病理改变并能改善IBS-D小鼠胆汁酸异常变化,这可能是由于柴参解郁汤修复肝肠循环胆汁酸合成和转化途径,增加结合型胆汁酸含量而起到保护肠道的作用.

The present study developed an ultra-fast liquid chromatography coupled with triple quadrupole-linear ion trap composite mass spectrometry(UHPLC-QTRAP-MS) to simultaneously determine the content of potential active components in Scutellariae Barbatae Herba and also to provide a reference approach for screening out the differential quality control components among different batches of Scutellariae Barbatae Herba. Chromatographic separations were conducted on a Thermo Acclaim~(TM) RSLC 120 C_(18) column(3.0 mm×100 mm, 2.2 μm) in a gradient program. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile, and the column temperature was maintained at 40 ℃. The flow rate was 0.4 mL·min~(-1) and the injection volume was 2 μL. The targeted compounds were monitored in the multiple reaction monitoring(MRM) mode. The acquired data were processed by hierarchical cluster analysis(HCA) and partial least square discriminant analysis(PLS-DA). Sixteen compounds all showed good linear relationship within the corresponding linear ranges and the R~2 values were all higher than 0.993 2. The RSDs of precision, repeatability, and stability were less than or equal to 3.7%. Mean recovery rates were in the range of 95.67% and 104.8% with RSDs≤3.2%. According to HCA and PLS-DA, all samples were clustered into four categories. Scutellarin, acteoside, scutellarein, and scutebarbatine X(VIP&gt;1) were considered as differential chemical markers in the four categories. In conclusion, the developed method can be used for the simulta-neous determination of the multiple components and quality control of Scutellariae Barbatae Herba.

As a critical regulator of bone resorption. osteoclastogenesis is closely associated with osteoporosis (OP) and commonly induced by receptor activator of nuclear factor-κB ligand (RANKL), suggesting that suppression of inflammation may improve OP. Urolithin A (UroA), an active metabolite of ellagic acid, is known to exert anti-inflammatory and antioxidative effects. However, whether UroA attenuates osteoclastogenesis remains unclear. Using a lipopolysaccharide (LPS)-induced bone loss model, we evaluated the effects of UroA on inflammatory osteoclastogenesis in mice and explored the potential mechanism from RANKL-related signaling pathway. UroA significantly improved LPS-induced bone loss and rescued the imbalance in bone microarchitecture parameters. Hematoxylin&eosin (H&E) and tartrate resistant acid phosphatase (TRAP) staining of femurs showed that UroA suppressed LPS-induced osteoclastogenesis accompanied by the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling. In RANKL-triggered mouse bone marrow-derived macrophages (BMDMs), UroA inhibited the formation of osteoclasts and Fibrous actin rings (F-actin rings), and decreased TRAP activity. Moreover, UroA significantly decreased mRNA and protein expression of major inflammatory cytokines in LPS-challenged RAW264.7 cells by decreasing the phosphorylation of NF-κB p65, c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase1/2 (Erk1/2), and p38. Furthermore, UroA may activate the Nrf2 signaling pathway by increasing mRNA and protein expression of antioxidant proteins. We conclude that UroA attenuated RANKL-induced osteoclastogenesis by suppressing the p38 mitogen-activated protein kinase (MAPK) pathway and inducing Nrf2 nuclear translocation. Thus, supplementation with UroA may help alleviate inflammation-induced bone loss and bone resorption.

Agrimonia pilosa is a perennial herbaceous flowering plant, commonly known as agrimony or hairy agrimony. The dried aerial parts of this species have been widely used for the treatment of acute diarrhea, hemostasis, and other inflammation-related diseases. However, information on the in vivo metabolism of A. pilosa constituents is limited. In this study, the phytochemical profile of A. pilosa was investigated using HPLC-Q-TOF-MS/MS combined with a nontargeted diagnostic ion network analysis strategy. An information-dependent acquisition method with multiple filters was utilized to screen possible prototypes and metabolites in complex biological matrices. Furthermore, various data-processing techniques were applied to analyze possible prototypes and their metabolites in rat plasma, feces, and urine following oral administration of A. pilosa extract. A total of 62 compounds, which belonged to five main structural classes (21 phenols, 22 flavonoids, 6 coumarins, 3 triterpenes, and 10 organic acids), were tentatively identified in A. pilosa. In addition, using our proposed stepwise method, 32 prototypes and 69 metabolites were detected in rat plasma, feces, and urine. The main metabolic pathways after the oral administration of A. pilosa extract were revealed to include methylation, dihydroxylation, demethylation, hydrolysis, sulfation, and glucuronidation. This comprehensive in vivo and in vitro identification of the possible active components in A. pilosa could provide a basis for understanding its various pharmacological activities.

Objective: The study aims to investigate the pharmacokinetic profile of Praeruptorin A and khellactone and in vitro hydrolysis of praeruptorin A to khellactone in different biological samples. Methods: A LC-MS/MS method was established. Analytes and internal standard (IS) were isolated using the protein precipitation method and then separated on a Thermo BDS Hypersil C18 (2.1 mm×50 mm, 2.4μm) column using a mobile phase consisting of 0.05% formic acid solution and acetonitrile. Samples were analyzed in positive electrospray-ionization (ESI) mode using multiple reaction monitoring (MRM). Results: The calibration plots gave desirable linearity (r2>0.99) in the concentration range from 0.99-990.0 and 2.0-2000.0 ng/mL for Praeruptorin A and khellactone, respectively. In addition, the LOQs of these analytes were sufficient for vivo pharmacokinetic study and vitro hydrolysis study of Praeruptorin A. The intra-batch and inter-batch precision were all within 14.05%, and the accuracy was between 89.39% and 109.50%. The extraction efficiency of PA and khellactone ranged from 76.35 ~ 89.58%. The matrix effects of analytes and the IS were between 89.67% ~ 105.26%. Conclusion: The liver CYPs mediated by the metabolism of PA may contribute to the systemic exposure of its active metabolite, khellactone, in rats.

Gut microbiota-derived tryptophan (TRP) metabolites, especially the indole derivatives, function as critical modulators of intestinal immune function and integrity via modulating the aryl hydrocarbon receptor pathway. Selective enrichment of these indole metabolites in biological samples is important for quantitative determination by routine methods such as HPLC. Here, we report a molecularly imprinted polymer (MIP) for solid-phase extraction (SPE) of TRP-derived indole metabolites from faeces of mice. The MIP was synthesized by surface polymerization by using indole-3-acetic acid (IAA) and acrylamide after cross-linking by ethylene glycol dimethacrylate (EGDMA) and initialed by 2',2-azobisisobutyronitrile (AIBN). The MIPs were then characterized by transmission electron microscope (TEM) and fourier transform infrared spectroscopy (FTIR), and the adsorption capacity, selectivity and reusability of MIPs were systematically evaluated. Results showed that IAA-MIPs showed a uniformly distributed nanoshell layer with a thin shell thickness of 26 nm. The IAA-MIPs could selectively adsorb IAA, indole-3-propionic acid (IPA), and indole-3-lactic acid (ILA) in a mixed solution that also contains TRP and tyrosine. The adsorption capacity of IAA-MIPs only slightly decreased with the increase of recycling use. As purification material for SPE, the IAA-imprinted polymers (IAA-MIPs) were successfully applied to extract IAA, IPA, and ILA from normal and colitis mice for HPLC determination. Collectively, these surface molecularly imprinted polymers could find extensive use to selective enrichment of microbiota-derived indole metabolites in biological samples.

肠道菌群是人体内种类最多、密度最大的微生物群落的总称.肠道菌群可通过与宿主共代谢,调控宿主多种生理功能、维持体内免疫平衡,进而维护人体健康.近年来的研究结果表明,肠道菌群与宿主的骨代谢密切相关.在多种病理状态或药物等引起肠道菌群改变后,可引起继发性的病理性骨丢失.随着高通量测序、基因敲除、无菌鼠繁殖等新技术的发展,越来越多的证据表明肠道菌群可通过直接调控,或通过内源性物质代谢调控,或改变与骨代谢相关的激素水平影响宿主的骨代谢.拟在全面阐述肠道菌群与骨代谢关系的基础上,总结肠道菌群调控骨代谢的潜在途径和常见中药的作用机制,并探讨其对主要骨代谢异常相关疾病在临床治疗和药物靶点研究中的启示.

目的 采用超高效液相色谱和-四级杆串联飞行时间质谱(UHPLC-Q-TOF/MS)法分析地榆水提物在大鼠血清、尿液及粪便中的原型成分及代谢产物,探讨地榆中活性成分在大鼠体内的代谢途径.方法 采用Thermo AcclaimTM RSLC 120 C18色谱柱(3.0 mm×100 mm,2.2 μm),以0.1％甲酸水溶液(A)-乙腈(B)为流动相进行梯度洗脱,流速0.4mL·min-1,进样量3μL,柱温40℃.运用电喷雾离子源(ESI),扫描范围m/z 50～1500,正、负离子模式采集数据.通过比较灌胃地榆水提物后大鼠血清、尿液和粪便样品和空白组样品的差异,对原型成分及其代谢产物进行鉴定.结果 灌胃给予地榆水提物后,从大鼠血清、尿液及粪便中鉴定出69个外源性成分,包括24个原型成分和45个代谢产物.其中24个原型成分包括16个酚类成分、7个三萜类成分及1个单萜烯醇苷.地榆水提物在大鼠体内的代谢途径主要包括还原、甲基化、脱水、氧化、硫酸化及葡萄糖醛酸结合等.结论 所鉴定出的原型成分和代谢产物可为进一步解析地榆体内代谢过程,阐明其药效物质基础提供实验依据.

目的 研究半枝莲提取物经肠道菌转化前后主要化学成分含量的变化及其对CYP1A1酶抑制能力的影响.方法 采用超高效液相色谱串联三重四级杆质谱(UPLC-Q-TRAP-MS)技术检测半枝莲提取液在肠道菌中温孵0、0.5、1、2、4、6、12、24h后主要活性成分的经时变化过程;通过CYP1A1酶活性分析实验研究半枝莲提取液经肠道菌转化不同时间点后对CYP1A1酶抑制作用的变化,并进一步研究半枝莲中主要黄酮类成分,野黄芩素、木犀草素、汉黄芩素及其苷对CYP1A1酶的抑制作用;利用分子对接验证野黄芩素、木犀草素、汉黄芩素与CYP1A1之间的结合情况.结果 半枝莲提取液经过肠道菌转化后,野黄芩素含量先增加后减少,木犀草素、汉黄芩素含量增加,黄酮苷类成分野黄芩苷、木犀草苷、汉黄芩苷含量随时间延长显著减少,而二萜类成分半枝莲碱A、半枝莲碱B、半枝莲碱X含量无明显变化;半枝莲提取液经肠道菌温孵2、4、6h后对CYP1A1酶活性的抑制作用增强,而12h后这种增强作用逐渐减弱;野黄芩素、木犀草素、汉黄芩素与CYP1A1蛋白对接后的结合自由能分别为-6.33、-6.61、-7.09kcal/mol,配体与CYP1A1中Phe224形成的π-π堆积作用为主要相互作用.结论 半枝莲中的黄酮类化合物是其发挥抑制CYP1A1酶活作用的主要活性物质,可能与半枝莲的清热解毒功效有关.

目的:运用网络药理学技术筛选葛根芩连汤治疗溃疡性结肠炎(ulcerative colitis,UC)的主要活性成分,预测活性成分的作用靶点,建立单味药-活性成分-作用靶点网络,进一步探讨葛根芩连汤治疗溃疡性结肠炎的潜在效应机制.方法:利用中药系统药理学技术平台(traditional chinese medicine systems pharmacology database and analysis platform,TCMSP)收集葛根芩连汤中4味中药的化学成分,借助人类基因数据库(GeneCards)获取UC的相关靶点基因,将药物作用靶点和疾病相关靶标基因取交集得到该方治疗UC的关键作用靶点,使用String数据库对关键作用靶点构建蛋白质相互作用网络,并通过Cytoscape软件将结果可视化.借助DAVID和ClueGo对关键作用靶点进行基因本体(gene ontology,GO)分析和基因组百科全书(KEGG)通路富集.结果:共收集葛根芩连汤138个活性成分和27个关键作用靶点.其中,STAT3、TNF、CXCL8、IL1B、VEGFA、TP53、IL10、AKT1等关键基因主要通过调控NF-κB、IL-17、Toll样受体、MAPK、PI3K-Akt、Jak/STAT、TNF等信号通路,在细胞外泌体、质膜外侧、细胞核中发挥作用.结论:葛根芩连汤治疗溃疡性结肠炎是多靶点、多个信号通路共同调节的复杂过程,预测葛根芩连汤治疗溃疡性结肠炎的潜在机制,为深入研究葛根芩连汤治疗溃疡性结肠炎的作用机制提供理论依据与参考.

目的:运用网络药理学技术预测补中益气汤治疗糖尿病肾病的作用靶点和信号通路,进一步探讨补中益气汤防治糖尿病肾病的物质基础和潜在作用机制.方法:利用中药系统药理学技术平台(TCMSP:)获得补中益气汤的活性成分及其作用靶点,从GeneCards数据库收集与糖尿病肾病相关的靶标基因,将药物作用靶点和疾病相关靶标基因取交集得到该方治疗糖尿病肾病的关键作用靶点,使用String数据库对关键作用靶点构建蛋白质相互作用网络,并通过Cytoscape软件将结果可视化.借助DAVID和ClueGo对关键作用靶点进行基因本体论(gene ontology,GO)分析和KEGG富集.结果:该方法找出了补中益气汤182个活性成分和41个关键作用靶点其中DPP4,MAPK14,MAPK3,MAPK8,MMP2,MMP3,MPO,MTTP,NOS3等关键基因主要通过调控AGE-RAGE信号通路,Toll样受体信号通路,cAMP信号通路,白细胞介素17(IL-17)信号通路,Relaxin信号通路,在细胞外泌体,细胞间隙,细胞核中发挥作用.结论:通过网络药理学预测补中益气汤防治糖尿病肾病的关键靶点及其相关信号通路,推测该方剂对糖尿病肾病的潜在作用机制,可能主要参与抗炎、调节糖脂代谢及氧化应激等过程,为阐明补中益气汤对糖尿病肾病的作用机制提供了科学依据.

为了基于网络药理学方法研究防己黄芪汤防治2型糖尿病的潜在作用机制,采用中药系统药理学数据库及技术平台(TCMSP),按照口服生物利用度OB≥30％和类药性DL≥0.18筛选处方中的活性成分及其相关靶点,通过Uniprot数据库找出对应官方基因名称.利用疾病靶点相关数据库检索与2型糖尿病相关的靶点基因.将活性成分所对应的靶点基因和2型糖尿病相关的靶点基因进行交集获得两者的共同基因即为防己黄芪汤治疗2型糖尿病的关键靶点.使用Cytoscape软件构建中药-活性成分-关键靶点网络,根据度值进行筛选,寻找该方剂防治2型糖尿病的主要物质基础.利用DAV I D数据库及ClueGo插件对关键作用靶点进行GO注释分析和KEGG通路分析,从而探究防己黄芪汤干预2型糖尿病的生物过程及作用通路.结果显示,筛选得到防己黄芪汤122个活性成分及242个潜在作用靶点,其中,与2型糖尿病重复的83个关键靶点中STAT3,AKT1,TNF,JUN,TP53,MAPK14,MAPK1,MAPK8可能通过调控AGE-RAGE信号通路、MAPK信号通路、AMPK信号通路、FoXO信号通路发挥治疗作用.由此得出结论:通过网络药理学预测防己黄芪汤防治2型糖尿病的关键靶点及其相关信号通路,推测该方剂对2型糖尿病的潜在作用机制,大都和脂代谢的过程、氧化应激过程相关,为进一步阐明防己黄芪汤对2型糖尿病的作用机制提供了科学依据.